This proposal tests a novel and innovative hypothesis in Rheumatoid Arthritis (RA) pathobiology. Specifically, we examine the role of complement C4B gene copy number variation (CNV) on RA pathogenesis and its interaction with HLA-DRB1 alleles (e.g. the shared epitope) in influencing RA phenotype (disease severity, B cell hyperactivity). Complement C4 proteins (C4A, C4B) are functionally distinguished by the nature of the covalent linkage they form with their targets: amino (C4A) vs thioester (C4B). Among their many essential roles, the acidic C4A and the basic C4B are important for the clearance of immune complexes. A deficiency of C4A is a well-established risk factor for human SLE, but the physiologic impact of C4B deficiency is under-studied. A diploid genome of a human subject contains zero to four copies of the C4B gene; serum levels of C4B strongly correlate with the number of C4B genes. In a RA population at Dartmouth, we observed that C4B deficiency (0-1 copy) is present in 43% of the seropositive patients, 31% of seronegative RA patients and 20% of non-RA patients or healthy controls (OR from 2.5 to 2.8). We hypothesize a particular role for C4B relative to C4A in RA mediated by its ability to clear immune complexes made up of anti-citrullinated protein antibodies (ACPA) and citrullinated antigens. We propose that the presence of deiminated-Arginines (i.e. citrullines with decreased numbers of amino groups) in these complexes results in a reduced ability of C4A to contribute to their clearance. As a result, the ability of the C4B-thioester carbonyl group to form a covalent linkage with a hydroxyl group of substrates for disposal makes C4B of much greater importance than C4A in the clearance of ACPA-immune complexes. Thus, deficient ACPA-based immune complex clearance associated with C4B deficiency would particularly enhance and favor maturation of anti-ACPA responses, promoting both seropositive RA and B cell hyperactivity. Hypothesis: We propose that C4B deficiency influences RA disease pathogenesis independent of the shared epitope (Aim 1). In Aim 2, we propose that C4B deficiency shapes RA phenotype and B cell hyperactivity (serum CXCL13, IgG and autoantibody levels). PUBLIC HEALTH RELEVANCE: One of the ways humans differ from each other in their genetic makeup arises from the variations in how many copies they have of certain genes. We have determined that variations in the gene copy number of the complement C4 genes are associated with rheumatoid arthritis. We have also found that rheumatoid arthritis patients differ in their level of systemic B cell activation. We propose that understanding how these attributes lead to the development of rheumatoid arthritis as well as the shape its severity and response to treatment are of great importance to the public health, since this disorder affects 1% of the population worldwide.